Mutant luciferases

ABSTRACT

Proteins are provided having luciferase activity with lower K m  than wild-type luciferases by altering the amino acid residue at position 270 of the wild-type to an amino acid other than glutamate. Greater heat stability than wild-type luciferases while retaining the lower K m  is provided by also replacing the glutamate equivalent to that at position 354 of  Photinus pyralis  luciferase or 356 of Luciola luciferases with an alternative amino acid, particularly lysine and/or the amino acid residue at 215 of  Photinus pyralis  and 217 of the Luciola species with a hydrophobic amino acid. DNA, vectors and cells that encode for and express the proteins are also provided as are test kits and reagents for carrying out luminescence assays using the proteins of the invention.

The present invention relates to novel proteins having luciferaseactivity and to DNA and vectors encoding for their expression.Particularly the present invention provides luciferases having lowerK_(m) for the substrate ATP than existing native and recombinantluciferases of wild and altered wild type.

Firefly luciferase catalyses the oxidation of luciferin in the presenceof ATP, Mg²⁺ and molecular oxygen with the resultant production oflight. This reaction has a quantum yield of about 0.88 (see DeLuca &McElroy (1978) and Seliger & McElroy (1960)) and this light emittingproperty has led to its use in luminometric assays where ATP levels arebeing measured.

Luciferase is obtainable directly from bodies of fireflies or byexpression from microorganisms including recombinant DNA constructsencoding for the enzyme. Significant species from which the enzyme maybe obtained, or DNA encoding for it derived, are the Japanese GENJI andHEIKE fireflies Luciola cruciata and Luciola lateralis, the EastEuropean Firefly Luciola mingrelica, the North American firefly(Photinus pyralis) and the glow-worm and the European glow-worm Lampyrisnoctiluca.

The heat stability of wild and recombinant type luciferases is such thatthey lose activity quite rapidly when exposed to temperatures in excessof about 30° C., particularly over 35° C., and this renders the enzymedeficient when used at high ambient temperatures. It is known thatJapanese firefly luciferase can be heat stabilised by mutating it at itsposition 217 to replace a threonine residue by an isoleucine residue(Kajiyama and Nakano (1993) Biochemistry 32 page 13795 to 13799); pHstability and specific activity also being increased.

Copending patent application GB 9405750.2 discloses an amino acidsubstitution that is capable of increasing the thermostability of interalia, Photinus pyralis which can be used with the change at 217 toprovide luciferase that is relatively heat stable at 50° C. or more.

The present invention relates to a further enhancement of the propertiesof luciferase enzymes, making them suitable for use in assays based uponthe detection of adenosine triphosphate at relatively low levels. Thisenhancement is provided by changing the amino acid at the positioncorresponding to position 270 in the Photinus pyralis luciferase aminoacid sequence whereby the Michaelis-Menten constant (K_(m)) of theenzyme is decreased as compared to a corresponding luciferase havingwild-type sequence. This corresponds to amino acid 272 in Luciolamingrelica, Luciola cruciata and Luciola lateralis. It also correspondsto amino acid 270 in Lampris Noctiluca.

The present enhancement further provides luciferases that arecharacterised by the ability to oxidise D-luciferin with light emissionof a different wavelength to that of wild-type luciferase, thus allowingthem to be used as specific labels in binding assays wherein thewavelength of light emitted is characteristic of a particular labelledmaterial being present, or allows DNA encoding for the luciferases to beused as a reporter DNA for genetically engineered cells and cellsderived therefrom.

Thus in the first aspect of the present invention there is provided aprotein having luciferase activity and having over 60% homology of aminoacid sequence with that of Photinus pyralis, Luciola mingrelica, Luciolacruciata or Luciola lateralis characterised in that the amino acidresidue corresponding to residue 270 of Photinus pyralis luciferase andresidue 272 of Luciola mingrelica, Luciola cruciata and Luciolalateralis luciferase is an amino acid other than glutamate. Preferablyis characterised in that it comprises a conserved amino acid sequenceF(1)XE(2)FL wherein (1) is D or E, (2) is T or L and X is the amino acidother than glutamate; F, E, L, D and T each relating to thecorresponding amino acid as provided for by the single letter amino acidcode.

The preferred amino acid X so far determined is lysine, or an analogueor modification thereof. Other preferred amino acids include arginine,glutamine and alanine.

In still more preferred forms of the present invention the protein ofthe invention also has the amino acid at the position corresponding toamino acid 217 of the Luciola firefly luciferases or 215 of Photinuspyralis changed to a hydrophobic amino acid, preferably to isoleucine,leucine or valine or analogue or these and/or has the amino acid at theposition corresponding to amino acid 356 of the Luciola fireflyluciferase or 354 of Photinus pyralis changed to an amino acid otherthan glutamate, particularly to lysine, arginine, leucine, isoleucine orhistidine or analogues or modifications of these.

In a second aspect of the invention there is provided DNA encoding forthe protein of the invention and in a third aspect there is provided avector, particularly a plasmid, comprising a luc gene (the gene encodingfor luciferase) in such a form as to be capable of expressing theprotein of the invention. Such forms are those where the vector includesDNA sequences capable of controlling the expression of the protein ofthe invention such that when incorporated into a microorganism host cellthe protein may readily be expressed as required, if necessary byaddition of suitable inducers.

The luc genes for Photinus pyralis, Luciola mingrelica, Luciola cruciataand Luciola lateralis are all known and isolatable by standard molecularbiology techniques. This is also the case for Lampris noctiluca.Photinus pyralis luc gene is commercially available from Promega as theplasmid pGEM-luc. Thus convenient methods and sources for derivingstarting material for production of DNA of the invention are (i) use ofnaturally occurring firefly genomic DNA and amplifying the luc gene fromit using eg, PCR, (ii) pGEM and (iii) pGLf37 plasmid of Kajiyama andNakano. Further genes encoding for proteins having luciferase activity,ie the activity of oxidising luciferin with the emission of light, willalso be suitable sources for starting material for obtaining a DNA, andultimately through gene expression, a protein of the invention.

Suitable vectors for use in manipulating wild type or other luc gene DNAin order to produce the DNA of the invention will be any vector in whichthe DNA can be contained within while alteration of the naturallyoccurring glutamate to an alternative amino acid is carried out. Forchemically induced mutagenesis, eg using agents such as hydroxylamine,this is not particularly critical and many suitable vectors will occurto those skilled in the art that will allow easy manipulation of thegene before and after the mutagenic process. It may be preferred tospecifically mutate the luc gene at the glutamate and thus a sitedirected mutagenesis operation will be required. Such operations may bemost easily carried out in vectors and these will be well known to thoseskilled in the art.

For expression of luc genes of wild and known type, and those of thepresent invention suitable vectors include pKK223-3, pDR540 (availablefrom Boehringer Mannheim) and pT7-7; the first two having the tacpromoter under control of the lactose repressor allowing expression tobe induced by presence of isopropyl-thiogalactoside (IPTG). pT7-7 allowscontrol by the T7-RNA polymerase promoter and thus provides the basisfor a very high level of gene expression in E. coli cells containing T7RNA polymerase. Of these vectors expression is found to be highest whenthe luc genes are inserted into the pT7-7 vector.

Expression of luciferase from a luc gene inserted into pKK223-3 andpDR540 results in the expression of wild-type N-terminal sequenceluciferase whilst expression from a luc gene inserted into pT7-7 resultsin synthesis of a fusion protein with extra N-terminal amino acidsA-R-I-Q (SEQ ID NO: 6). The ribosome binding site and start codon of theluc gene in each of the respective vectors with the luc gene present(named constructs pPW204, pPW116 and pPW304) are shown in Table 1 of theExamples. pPW601a referred to below is derived by removing the uniqueXho I site pPW116.

A third aspect of the present invention provides cells capable ofexpressing the proteins of the invention; methods for producing suchproteins using these cells and test kits and reagents comprising theproteins of the invention. Also provided are assay methods wherein ATPis measured using luciferin/luciferase reagents, as is well known in theart, characterised in that the luciferase is a protein of the invention.Luciferase preparations of the invention are relatively low in K_(m)with respect to the corresponding wild type and recombinant luciferases,and preferred double and triple change luciferases (ie 215; 270; 354changed Photinus or 217; 272; 356 changed Luciola, or 215; 270; 354changed L. noctiluca also have the property of relative thermostabilityat 30-70° C., particularly 37-60° C., and especially 40-50° C. Thus thepresent invention has been established as not preventing thethermostability enhancements of other contemporaneous and previous workby the present inventors and others from being used.

Any cell capable of expressing heterologous protein using DNA sequencesin its DNA, or in vectors such as plasmids contained in the cell, may beused to express the proteins of the invention. Typical of such cellswill be yeast and bacterial cells such as Saccharomyces cerevisiae andEscherichia coli cells, but many other host organisms suitable for thepurpose of protein expression will occur to those skilled in the art.

The protein may be expressed as a protein of similar structure to nativeand known recombinant luciferases, or may be expressed as a fusion orconjugate of such proteins with other amino acids peptides, proteins orother chemical entities, eg the A-R-I-Q (SEQ ID NO: 6) sequence above.

It will be realised by those skilled in the art that certain hosts mayhave particular codon preferences, eg bacteria in some cases usedifferent codons to yeast, and thus the DNA incorporated into such ahost may advantageously be altered to provide a degenerate codon for agiven amino acid that will give more favourable expression in that host.Such degenerate DNAs are of course included in the scope of the DNA ofthe invention.

E. coli BL21 (DE3) is one suitable host and has the T7 RNA polymeraseintegrated stably into its chromosome under control of the induciblelacUV5 promoter and is thus compatible with pT7-7 derived constructs.

E. coli B strains like BL21 lack the lon protease and the ompT outermembrane protease. These deficiencies can help to stabilise theexpression and accumulation of foreign proteins in E. coli. Assays ofcrude extracts of E. coli BL21 (DE3) containing each of the threeexpression constructs described above indicated that the highest levelsof expression of luciferase were obtained from cells containing theconstruct pPW304 (see Table 2). Other suitable cell lines, such as thatof the E. coli JM109 cells used in the Examples below will occur tothose skilled in the art.

The proteins, DNA, vectors and cells of the invention will now bedescribed by way of illustration only by reference to the followingnon-limiting Examples, Figures, Tables and Sequence listing. Furtherproteins, conjugates of proteins, DNA, vectors and cells, and assays andtest kits incorporating any of the above will occur to those skilled inthe art in the light of these.

FIGURES

FIG. 1: shows a restriction map of plasmid pPW204 derived from pKK223-3by insertion of a luc gene as described in the Examples below.

FIG. 2: shows a restriction map of plasmid pPW116 derived from pDR540 byinsertion of a luc gene as described in the Examples below.

FIG. 3: shows a restriction map of plasmid pPW304 derived from pT7-7 byinsertion of a luc gene as described in the Examples below.

FIG. 4: shows a restriction map of plasmid pPW601a derived from pDR540and BamH1/Sst1 fragment from pGEM-luc with the Xho site removed.

FIG. 5: shows a graph of heat inactivation of recombinant wild-typePhotinus luciferases (Sigma), K_(m) changed luciferase of the invention,the thermostable 354 lysine mutant provided by copending GB 9405750.2and K_(m)/354 lysine double mutant of the present invention incubated ata given temperature for 16 minutes as described in the Examples below.

FIG. 6: shows a restriction map of pT7-7 after Tabor (SEQ ID NO: 12 andSEQ ID NO: 13).

SEQUENCE LISTING

The sequence listing provided at the end of this specification describesDNA and amino acid sequences as follows:

SEQ ID No 1: shows the DNA sequence of a DNA encoding for luciferase ofthe invention wherein the Photinus pyralis wild-type codon at 811 to 813is mutated; for lysine only the base at 811 is mutated to an A. It alsoshows the position for introducing thermostability at 1063-65.

SEQ ID No 2: shows the amino acid sequence of a protein of the inventionwherein the Photinus pyralis wild-type amino acid 270 glutamate has beenchanged to a residue Xaa other than glutamate.

SEQ ID No 3: shows the sequence of the oligonucleotide used for the SDMmutation of pPW601a to give a lysine instead of glutamate at position270.

SEQ ID No 4: shows the amino acid sequence of a protein of the inventionwherein the Photinus pyralis wild-type amino acid 270 glutamate has beenchanged to lysine and the 354 amino acid changed to lysine.

SEQ ID No 5: shows the sequence of the oligonucleotide used for the SDMmutation of pPW601a to give a lysine instead of glutamate at position354.

EXAMPLES Example 1: Production of Plasmids Containing DNA of theInvention

Plasmids pKK223-3 and pDR540 were obtained form Boehringer Mannheim;pDR540 is also available from Pharmacia Biotech St Albans UK. PhagemidpBluescript II SK(+) was obtained from Stratagene La Jolla, USA. E. colistrain BL21 (DE3) was used for the expression of luciferase from pT7-7derived plasmids and E. coli strain JM109 (4) was used in all cloningexperiments and for the expression of luciferase from pDR540 derivedplasmids.

Plasmid pT7-7 (see Current protocols in Molecular Biology Vol II Section16.2.1) was obtained from Stan Tabor, Dept of Biol Chem, Harvard MedicalSchool, Boston, Mass. 02115 and (as shown in FIG. 6) contains T7 RNApolymerase promoter Ø10 and the translation start site for the T7 gene10 protein (T7 bp 22857 to 22972) inserted between the PvuII and ClaIsites of pT7-5. Unique restriction sites for creation of fusion proteins(after filling in 5′ ends) are Frame 0: EcoR1; Frame 1: NdcI, SmaI,ClaI; Frame 2: BamHI, SalI, HindIII. SacI site of the originalpolylinker is removed by deletion and an additional XbaI site isprovided upstream of the start codon.

As stated in the preamble to the Figures, pPW204 was derived frompKK223-3; pPW116 was derived from pDR540; pPW304 derived from pT7-7;each by insertion of a luc gene derived from Promega pGEM-luc usingstandard restriction endonuclease and ligation techniques while pPW601was created by cloning the luc gene and BamH1/Sst1 fragment frompGEM-luc into pDR540 and pPW601a as derived by removing the unique Xho Isite in the polylinker of the plasmid. pPW601a contains a uniquerecognition site for Ava I which simplifies the SDM procedure forluciferase amino acid 354 changes.

For production of pPW304, pT7-7 is digested with EcoRI, the ends filledusing Klenow fragment, the product digested with SalI and the DNA gelpurified; pGEM-luc is digested with Bam HI, the overhangs produceddigested with MBN, the product digested with SalI and the 1 Kb fragmentproduced purified and ligated to the purified pT7-7 DNA.

Transformation of plasmids into BMH 71-18 mut S cells was carried outusing a Bio-Rad Gene Pulser version 2-89. For production of pPW601clones harvested cells and purified mixed plasmid pool containingmutated and parental plasmids were provided and secondary restrictiondigest with AvaI was carried out before transformation into E. coliJM109 cells. These cells were plated on selective media (LB agar+50μg/ml ampicillin) and clones screened by purifying their plasmid DNA andanalysing for the desired change. Plasmid DNA was purified usingalkaline lysis (Birnboim & Doly (1979) Nucleic Acids Research 7, p1513).

Relative levels of expression of luciferase from each of the constructspPW204, pPW116 and pPW304 are 0.1:0.5:1.0 from E. coli BL21 (DE3). Cellswere grown in LB at 37° C. to an OD 600 of 0.3 then induced with IPTGand growth allowed to continue for 4 hours after which crude extract wasprepared and luciferase activity measured.

TABLE 1 Ribosome binding sites (underlined) and start codons inthe expression constructs pPW304(SEQ ID NO:7)AAGGAGATATACAT ATG* CGT AGA ATT CAA ATG pPW116(SEQ ID NO:8)AGGAAACAGGATCCA ATG* pPW204(SEQ ID NO:9) AGGAAACAGCAA ATG*

Partial purification of luciferases was carried out on E. coli JM109cells harvested in the early stationary phase then resuspended in 50 mMTris HCl pH 8.0 containing 50 mM KCl, 0.5 mM dithiothreitol and 1 mMEDTA (Buffer A). Cells were broken up by disruption in a MSE soniprep(amplitude 14μ) and the lysate centrifuged at 30000×g for 1 hour. Thesupernatant of the crude extract was then subjected to fractionationwith ammonium sulphate and the fraction precipitated between 35% and 55%saturation contained luciferase activity and was dissolved in Buffer Aand dialysed overnight against 500 ml of 50 mM Tris-HCl buffer pH8.0containing 0.4 mM DTT (Buffer B).

Full purification of luciferases was carried out by applying theprecipitated and dialysed enzyme to a Mono Q (HR10/10) anion exchangecolumn and eluting that with a linear gradient of 0-200 mM NaCl inBuffer B (flow-rate 4 ml/minute; 2 ml fractions). Peak fractionscontaining luciferase activity were made to 50% glycerol (v/v) andstored at −20° C.

Firefly luciferase (prepared from a crystalline suspension, Cat NoL9009) and coenzyme A and ATP were obtained from Sigma Chemical Co.Beetle luciferin potassium salt was obtained from Promega Corporation,Madison Wis., USA. Cell extracts were prepared as described in thePromega technical bulletin No 101. Aliquots of E. coli cultures werelysed in cell culture lysis reagent (25 mM Tris-phosphate, pH7.8, 2 mMDTT, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2.5 mg/ml BSA, 1.25 mg/mllysozyme) for 10 minutes at room temperature, centrifuged at 16000 g for2 minutes and then stored on ice prior to assay.

Luciferase activity of cell lines was assayed by monitoringbioluminescence emitted by colonies by transferring these to nylonfilters (Hybond N, Amersham) and then soaking the filters with 0.5 mMluciferin in 100 mM sodium citrate buffer pH5.0 (Wood & DeLuca, (1987)Anal Biochem 161 p501-507) at room temperature. Luciferase assays invitro were performed at 21° C. using 100 μl of assay buffer (20 mMTricine pH7.8 containing 1 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 0.27 mMconenzyme A, 0.47 mM D-luciferin, 0.53 mM ATP and 1 to 5 μl of sample).The final pH of the assay cocktail was 7.8 and light measurements weremade with a BioOrbit 1250 luminometer or in microtitre plates using alabsystems luminoskan RS plate luminometer.

Protein was determined by the method of Bradford (1976) Anal. Biochem.72 p248-254 using BSA as standard. For production of non-specificchemical mutations of DNA, plasmids containing luc genes were treatedaccording to the method of Kironde et al (1989) Biochem. J. 259,p421-426 using 0.8M hydroxylamine, 1 mM EDTA in 0.1 mM sodium phosphatepH6.0 for 2 hours at 65° C.

The K_(m) mutant was initially generated by hydroxylamine inducedmutagenesis of the luc gene within pPW304 to provide plasmid 304 G1bearing a single base change in the DNA sequence at 811 of SEQ ID No 1resulting in an amino acid glutamate change to lysine at position 270. A1.1 kb DNA fragment (BstE II/Stu I) was cloned from pPW304 and used toreplace the corresponding fragment in pP601a to form pPW601G1, thusproviding a luc gene encoding for luciferase without the four extraamino acids encoded by pPW304 (M not included from M-A-R-I-Q).

This mutagenised plasmid was desalted on a G60 DNA grade Nick column(Pharmacia) followed by transformation into E. coli BL21 (DE3).Luciferase expressed from this showed an identical low K_(m) phenotypeto that of the original mutant.

Double stranded DNA sequencing was performed by the dideoxy chaintermination method of Sanger et al (1977) Proc. Nat. Acad. Sci. (USA)74, 5463-5467 using [alpha-³²P]dATP and electrophoresis in 8M urea (6%wt/vol) polyacrylamide gels. Automatic sequencing was also undertakenusing a DNA model 373A automated sequencer (Applied Biosystems).

Assay for determining the K_(m) value of this luciferase with respect toATP was carried out at 21° C. with 100 μl of assay buffer (20 mM tricinepH7.8 containing 1.0 mM MgSO₄, 0.1 mM EDTA, 33 mM dithiothreitol, 270 μMcoenzyme A, 470 μM D-luciferin and 6.25 to 400 μM ATP) using aluminometer to measure cpm.

The K_(m) value for 601a-recombinant wild-type was determined to be 66.1μM (s.e. 4.1); for 601aK (thermostable mutant 354 lysine) was 61.3 (s.e.4.7) and for 601aG1 (270 lysine K_(m) change) was 28.7 (s.e. 0.9) thusillustrating that the 270 change more than halves the ATP concentrationfor which the enzyme is optimised.

The effect of the 270 change on the thermostability of luciferase isnegative, with t_(½) activity being reached after only 2 minutes ascompared to wild-type at 7 minutes, both at 37° C.; however at 30° C.the specific activity of 270 is greater than wild-type.

Example 2: Preparation of ‘double mutant’ 270K: 354K Photinus pyralisluciferase

In order to offset the reduced thermostability of the 270 changeluciferase, a double change luciferase was provided by using sitedirected mutagenesis to engineer the lysine change at 354 into the270-lysine luciferases encoding DNA and plasmid described in Example 1.This involved mutation using specifically designed oligonucleotides toconvert pPW601aG1 to pPW601a to G1K.

The oligonucleotide used to generate the 354 lysine change by SDM wasCATCCCCCTTGGGTGTAATCAG (SEQ ID No 5) with the underlined T being themismatch.

The site directed mutagenesis required to convert the glutamate 354 ofpPW601aE270K, and where required for direct synthesis of 270 mutant frompPW601a, to desired amino acids is carried out using the followingprotocol with oligonucleotides designed as required.

Site Directed Mutagenesis Protocol: Plasmid selected is denatured andannealed with selection and mutagenic oligonucleotides for the desiredchange. The mutant DNA strand is synthesised and ligated and the wholeprimary restriction digested with a restriction endonuclease.Oligonucleotide primers for sequencing and SDM were synthesised using anApplied Biosystems model 380A DNA synthesiser. DNA oligonucleotideprimers were designed to destroy either a unique Ava I site within theluc gene or the unique ScaI site within the gene for β-lactamase; thepresence of these sites being used to select against plasmids that hadnot undergone mutagenesis. Precise protocols were as described in theTransformer™ Site-Directed Mutagenesis Kit (Version 2.0) sold byClontech Laboratories Inc (US) catalog No K1600-1.

The restriction map for pPW601 derived from pDR540 and cloned luc geneis shown as FIG. 4. Site directed mutagenesis was carried out asdescribed above and in the Clontech instructions such as to convert thewild-type Photinus luc gene inserted therein into a sequence as shown inSEQ ID No 1 with expressed protein of amino acid sequence modified atposition 270 as shown as Xaa in SEQ ID No 2 to Lysine.

K_(m) studies were carried out as described in Example 1 while heatinactivation studies were carried out using crude extracts at 37° C. inlysis buffer (25 mM Tris phosphate pH7.8, 2 mM DTT. 2 mM EDTA, 10%glycerol and 1% Triton X-100) at various time points aliquots of enzymewere removed and assayed as described above (with 530 μM ATP). Theremaining activity was plotted against time.

The K_(m) value for 601aG1K, the double change of this example, wasfound to be 25.2 μM (s.e. 1.5) being again less than half that of thecorresponding 354 lysine mutant and the wild-type luciferase.

The t_(½) value, the time after which the activity of the luciferase isreduced on continuous heating to 50% of its initial value, was found tobe as follows:

601a (recombinant wild-type) t_(½)reached after 7.0 minutes 601aG1 (270K_(m) change) t_(½)reached after 1.75 minutes 601aK (354 thermostablechange) t_(½)reached after >35 minutes 601aG1K (270 + 354 change)t_(½)reached after 10.5 minutes

The above data is included below (plus other data) along with Km values.

Km ATP t_(½)37° C. (min) recombinant wild type 66.1 7.0 E270K 28.7 1.75E354K 61.3 >35 E270K + E354K 25.2 10.5 E270R 32.0 1.75 E270Q 44.0 1.75E270A 37.0 1.75

13 1722 base pairs nucleic acid double unknown cDNA to mRNA NO NOPhotinus pyralis CDS 4..1653 misc_difference replace(811..813, “”) 1CAAATGGAAG ACGCCAAAAA CATAAAGAAA GGCCCGGCGC CATTCTATCC TCTAGAGGAT 60GGAACCGCTG GAGAGCAACT GCATAAGGCT ATGAAGAGAT ACGCCCTGGT TCCTGGAACA 120ATTGCTTTTA CAGATGCACA TATCGAGGTG AACATCACGT ACGCGGAATA CTTCGAAATG 180TCCGTTCGGT TGGCAGAAGC TATGAAACGA TATGGGCTGA ATACAAATCA CAGAATCGTC 240GTATGCAGTG AAAACTCTCT TCAATTCTTT ATGCCGGTGT TGGGCGCGTT ATTTATCGGA 300GTTGCAGTTG CGCCCGCGAA CGACATTTAT AATGAACGTG AATTGCTCAA CAGTATGAAC 360ATTTCGCAGC CTACCGTAGT GTTTGTTTCC AAAAAGGGGT TGCAAAAAAT TTTGAACGTG 420CAAAAAAAAT TACCAATAAT CCAGAAAATT ATTATCATGG ATTCTAAAAC GGATTACCAG 480GGATTTCAGT CGATGTACAC GTTCGTCACA TCTCATCTAC CTCCCGGTTT TAATGAATAC 540GATTTTGTAC CAGAGTCCTT TGATCGTGAC AAAACAATTG CACTGATAAT GAATTCCTCT 600GGATCTACTG GGTTACCTAA GGGTGTGGCC CTTCCGCATA GAACTGCCTG CGTCAGATTC 660TCGCATGCCA GAGATCCTAT TTTTGGCAAT CAAATCATTC CGGATACTGC GATTTTAAGT 720GTTGTTCCAT TCCATCACGG TTTTGGAATG TTTACTACAC TCGGATATTT GATATGTGGA 780TTTCGAGTCG TCTTAATGTA TAGATTTGAA NNNGAGCTGT TTTTACGATC CCTTCAGGAT 840TACAAAATTC AAAGTGCGTT GCTAGTACCA ACCCTATTTT CATTCTTCGC CAAAAGCACT 900CTGATTGACA AATACGATTT ATCTAATTTA CACGAAATTG CTTCTGGGGG CGCACCTCTT 960TCGAAAGAAG TCGGGGAAGC GGTTGCAAAA CGCTTCCATC TTCCAGGGAT ACGACAAGGA 1020TATGGGCTCA CTGAGACTAC ATCAGCTATT CTGATTACAC CCNNNGGGGA TGATAAACCG 1080GGCGCGGTCG GTAAAGTTGT TCCATTTTTT GAAGCGAAGG TTGTGGATCT GGATACCGGG 1140AAAACGCTGG GCGTTAATCA GAGAGGCGAA TTATGTGTCA GAGGACCTAT GATTATGTCC 1200GGTTATGTAA ACAATCCGGA AGCGACCAAC GCCTTGATTG ACAAGGATGG ATGGCTACAT 1260TCTGGAGACA TAGCTTACTG GGACGAAGAC GAACACTTCT TCATAGTTGA CCGCTTGAAG 1320TCTTTAATTA AATACAAAGG ATATCAGGTG GCCCCCGCTG AATTGGAATC GATATTGTTA 1380CAACACCCCA ACATCTTCGA CGCGGGCGTG GCAGGTCTTC CCGACGATGA CGCCGGTGAA 1440CTTCCCGCCG CCGTTGTTGT TTTGGAGCAC GGAAAGACGA TGACGGAAAA AGAGATCGTG 1500GATTACGTCG CCAGTCAAGT AACAACCGCG AAAAAGTTGC GCGGAGGAGT TGTGTTTGTG 1560GACGAAGTAC CGAAAGGTCT TACCGGAAAA CTCGACGCAA GAAAAATCAG AGAGATCCTC 1620ATAAAGGCCA AGAAGGGCGG AAAGTCCAAA TTGTAAAATG TAACTGTATT CAGCGATGAC 1680GAAATTCTTA GCTATTGTAA TCCTCCGAGG CCTCGAGGTC GA 1722 550 amino acidsamino acid single unknown protein NO Photinus pyralis Modified-site 2702 Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 1015 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 2530 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 4045 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 5560 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 7075 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 8590 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg100 105 110 Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val PheVal 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys LysLeu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr AspTyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser HisLeu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser PheAsp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly SerThr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Ala CysVal Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln IleIle Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His HisGly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly PheArg Val Val Leu Met Tyr Arg Phe Glu Xaa Glu Leu 260 265 270 Phe Leu ArgSer Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro ThrLeu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 AspLeu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr340 345 350 Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val ProPhe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr LeuGly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met IleMet Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala LeuIle Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr TrpAsp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser LeuIle Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu SerIle Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala GlyLeu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val ValVal Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val AspTyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly GlyVal Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys LeuAsp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 GlyGly Lys Ser Lys Leu 545 550 30 base pairs nucleic acid single unknownDNA (genomic) NO Photinus pyralis misc_difference replace(10, “”) 3GTATAGATTT GAAAAAGAGC TGTTTTTACG 30 550 amino acids amino acid singleunknown protein NO Photinus pyralis Modified-site 354 Modified-site 2704 Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro 1 5 1015 Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg 20 2530 Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu 35 4045 Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala 50 5560 Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val 65 7075 80 Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu 8590 95 Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg100 105 110 Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val PheVal 115 120 125 Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys LysLeu Pro 130 135 140 Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr AspTyr Gln Gly 145 150 155 160 Phe Gln Ser Met Tyr Thr Phe Val Thr Ser HisLeu Pro Pro Gly Phe 165 170 175 Asn Glu Tyr Asp Phe Val Pro Glu Ser PheAsp Arg Asp Lys Thr Ile 180 185 190 Ala Leu Ile Met Asn Ser Ser Gly SerThr Gly Leu Pro Lys Gly Val 195 200 205 Ala Leu Pro His Arg Thr Leu CysVal Arg Phe Ser His Ala Arg Asp 210 215 220 Pro Ile Phe Gly Asn Gln IleIle Pro Asp Thr Ala Ile Leu Ser Val 225 230 235 240 Val Pro Phe His HisGly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu 245 250 255 Ile Cys Gly PheArg Val Val Leu Met Tyr Arg Phe Glu Lys Glu Leu 260 265 270 Phe Leu ArgSer Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val 275 280 285 Pro ThrLeu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr 290 295 300 AspLeu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser 305 310 315320 Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile 325330 335 Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr340 345 350 Pro Lys Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val ProPhe 355 360 365 Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr LeuGly Val 370 375 380 Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met IleMet Ser Gly 385 390 395 400 Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala LeuIle Asp Lys Asp Gly 405 410 415 Trp Leu His Ser Gly Asp Ile Ala Tyr TrpAsp Glu Asp Glu His Phe 420 425 430 Phe Ile Val Asp Arg Leu Lys Ser LeuIle Lys Tyr Lys Gly Tyr Gln 435 440 445 Val Ala Pro Ala Glu Leu Glu SerIle Leu Leu Gln His Pro Asn Ile 450 455 460 Phe Asp Ala Gly Val Ala GlyLeu Pro Asp Asp Asp Ala Gly Glu Leu 465 470 475 480 Pro Ala Ala Val ValVal Leu Glu His Gly Lys Thr Met Thr Glu Lys 485 490 495 Glu Ile Val AspTyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu 500 505 510 Arg Gly GlyVal Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly 515 520 525 Lys LeuAsp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys 530 535 540 GlyGly Lys Ser Lys Leu 545 550 22 base pairs nucleic acid single unknowncDNA to mRNA NO Photinus pyralis misc_difference replace(10, “”) 5CATCCCCCTT GGGTGTAATC AG 22 4 amino acids amino acid single linearpeptide unknown 6 Ala Arg Ile Gln 1 32 base pairs nucleic acid singlelinear DNA (genomic) unknown 7 AAGGAGATAT ACATATGCGT AGAATTCAAA TG 32 18base pairs nucleic acid single linear DNA (genomic) unknown 8 AGGAAACAGGATCCAATG 18 15 base pairs nucleic acid single linear DNA (genomic)unknown 9 AGGAAACAGC AAATG 15 7 amino acids amino acid single linearpeptide unknown 10 Phe Xaa Xaa Glu Xaa Phe Leu 1 5 10 amino acids aminoacid single linear peptide unknown 11 Thr Pro Xaa Gly Asp Asp Lys ProGly Ala 1 5 10 186 base pairs nucleic acid both both other nucleic acid/desc = “PLASMID” unknown CDS 124..186 12 CGATTCGAAC TTCTCGATTCGAACTTCTGA TAGACTTCGA AATTAATACG ACTCACTATA 60 GGGAGACCAC AACGGTTTCCCTCTAGAAAT AATTTTGTTT AACTTTAAGA AGGAGATATA 120 CAT ATG GCT AGA ATT CGCGCC CGG GGA TCC TCT ACA GTC GAC CTG CAG 168 Met Ala Arg Ile Arg Ala ArgGly Ser Ser Thr Val Asp Leu Gln 1 5 10 15 CCC AAG CTT ATC ATC GAT 186Pro Lys Leu Ile Ile Asp 20 21 amino acids amino acid linear proteinunknown 13 Met Ala Arg Ile Arg Ala Arg Gly Ser Ser Thr Val Asp Leu GlnPro 1 5 10 15 Lys Leu Ile Ile Asp 20

What is claimed is:
 1. An isolated mutant luciferase protein havingluciferase activity, which has over 60% amino acid sequence homology tothe luciferase from Photinus pyralis, Luciola mingrelica, Luciolacruciata or Luciola lateralis and which includes the amino acid sequenceF(1)XE(2)FL (SEQ ID NO: 6), where (1) is D or E, (2) is T or L and X isan amino acid other than glutamate and is at a position corresponding toamino acid residue 270 of Photinus pyralis luciferase as shown in SEQ IDNO:
 2. 2. A protein as claimed in claim 1 wherein it further comprisesan amino acid sequence TPXGDDKPGA (SEQ ID NO. 7) wherein X is an aminoacid residue other than glutamate.
 3. A protein as claimed in claim 1,wherein the amino acid residue X is lysine.
 4. A protein as claimed inclaim 1 wherein the amino acid residue corresponding to residue 215 ofPhotinus pyralis luciferase is a hydrophobic amino acid.
 5. A protein asclaimed in claim 4 wherein the residue corresponding to residue 215 ofPhotinus pyralis luciferase is one of isoleucine, leucine or valine. 6.A protein as claimed in claim 1 wherein the amino acid residuecorresponding to residue 354 of Photinus pyralis luciferase is an aminoacid other than glutamate.
 7. A protein of claim 6 wherein the residuecorresponding to residue 354 of Photinus pyralis luciferase is one oflysine, arginine, leucine, isoleucine or histidine.
 8. An isolated DNAencoding for a protein as claimed in claim
 1. 9. An isolated DNA asclaimed in claim 8 comprising a nucleotide sequence as described in SEQID No 1 wherein nucleotide residues 811-813 form a codon encoding anamino acid other than glutamate.
 10. An isolated DNA as claimed in claim9 wherein the codon encodes lysine.
 11. A vector comprising a luc geneencoding a protein as claimed in claim
 1. 12. A vector as claimed inclaim 11 obtainable by treating a vector containing a wild-type orrecombinant luc gene by site directed mutagenesis to change the codonresponsible for encoding the glutamate at position 270 of Photalispyralis luciferase to an alternative amino acid.
 13. A vector as claimedin claim 12 wherein the alternative amino acid is lysine.
 14. A vectoras claimed in claim 11 selected from pKK223-3, pDR540 and pT7-7 intowhich said luc gene has been ligated.
 15. A cell transformed with a DNAor a vector capable of expressing a protein as claimed in claim
 1. 16. Acell as claimed in claim 15 which is an E. coli or a S. cerevisiae cell.17. A test kit for performance of an assay through measurement of ATPwherein the kit comprises a protein as claimed in claim
 1. 18. An assaymethod wherein ATP is measured using luciferin and luciferase togenerate light the quantity of which is related to the amount of ATPwherein the luciferase is a protein as claimed in claim
 1. 19. An assaymethod as claimed in claim 18 wherein the assay is carried out at atemperature of from 30° C. to 70° C.
 20. An assay method as claimed inclaim 18 wherein the assay is carried out at a temperature of from 37°C. to 60° C.
 21. An assay method as claimed in claim 18 wherein theassay is carried out at a temperature of from 40° C. to 50° C.
 22. Aprotein comprising an amino acid sequence as described in SEQ ID No 2wherein Xaa is chosen from arginine, glutamine and alanine.
 23. A mutantluciferase protein of claim 1 wherein said luciferase protein is afirefly or a glow worm luciferase.
 24. A mutated luciferase of claim 1wherein said luciferase is a Photinus luciferase.
 25. A mutantluciferase protein as claimed in claim 1 wherein said luciferase has aK_(m) to the substrate ATP which is lower than that of the correspondingwild type luciferase.